Use of hepatitis b x-interacting protein (hbxip) in modulation of apoptosis

ABSTRACT

Novel methods of regulating cellular apoptosis by affecting the interaction of hepatitis B X-interacting protein (HBXIP) with Survivin are described. More specifically, these novel methods of enhancing apoptosis of neoplastic cells comprises inhibiting interaction of hepatitis B X-interacting protein (HBXIP) with Survivin using siRNA or antisense compounds.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/665,975 filed on Sep. 18, 2003, which claims the benefit ofpriority under 35 U.S.C. 119(e) of the U.S. Provisional Application60/412,109 filed Sep. 18, 2002, the disclosures of which are expresslyincorporated herein by reference in its entirety.

GOVERNMENTAL INTEREST

This invention was made with government support under grant numberAG15343 awarded by the National Institutes of Health. The United StatesGovernment may have certain rights in this invention.

FIELD OF THE INVENTION

The present invention is related to regulation of apoptosis. Morespecifically the present invention is related to the use of hepatitis BX-interacting protein (HBXIP) in the stimulation of apoptosis inneoplastic diseases.

BACKGROUND OF THE INVENTION

Survivin represents one of the most tumor-specific genes in the humangenome according to comparisons of the transcriptomes of normal andmalignant cells (Velculescu, V. E. et al. 1999 Nature Gen 23:387-388).The 17 kDa protein Survivin is scarcely expressed in normal adulttissues, but is found at high levels in most human cancers (Ambrosini,G. et al. 1997 Nature Med 3:917-921). Normally, Survivin is expressedonly during late stages of the cell cycle (particularly mitosis andanaphase), where it associates with the mitotic spindle and relatedstructures, performing functions important for chromosome segregationand cytokinesis (Li, F. et al. 1998 Nature 396:580-587; Reed, J. C. &Bischoff, J. R. 2000 Cell 102:545-548). Many cancers, however, containconstitutively high levels of cytosolic p17 Survivin, andover-expression of this protein has been shown to block apoptosis bothin vitro in cultured cells and in vivo in transgenic mice (Ambrosini, G.et al. 1997 Nature Med 3:917-921; Grossman, D. et al. 2001 J Invest108:991-999; Reed, J. C. 2001 J Clin Invest 108:965-969). Antisense anddominant-negative experiments have provided proof of concept evidencesuggesting that interfering with Survivin function could be a worthwhilestrategy for promoting apoptosis of tumor cells (Reed, J. C. 2001 J ClinInvest 108:965-969; Li, F. et al. 1999 Nature Cell Biol 1:461-466;Mesri, M., et al. 2001 J Clin Invest 108:981-990). However, at presentit is unclear how Survivin blocks apoptosis.

Survivin is a member of a family of proteins which all contain acharacteristic zinc-binding fold called the BIR domain. Many of theseBIR-containing proteins have been shown to suppress apoptosis whenover-expressed, thus prompting the term Inhibitor of Apoptosis Proteins(IAPs). The principal mechanism of apoptosis suppression by IAP-familymembers such as XIAP has been defined. These proteins directly bind andpotently suppress the activity of Caspases (Deveraux, Q. L. & Reed, J.C. 1999 Genes Dev 13:239-252), a group of intracellular proteasesresponsible for apoptosis (reviewed in Cryns, V. & Yuan, Y. 1999 GenesDev 12:1551-1570). Though some studies have suggested that p17 Survivinalso binds and suppresses Caspases, others have failed to demonstratedirect effects on these proteases (Shin, S. et al. 2001 Biochem40:1117-1123; Verdecia, M. A. et al. 2000 Nature Struct Biol 7:602-608;Conway, E. M. et al. 2000 Blood 95:1435-1442; Banks, D. P. et al. 2000Blood 96:4002-4003).

Because Survivin is over-expressed in the majority of tumors(Velculescu, V. E. et al. 1999 Nature Gen 23:387-388; Ambrosini, (G. etal. 1997 Nature Med 3:917-921), this protein has emerged as a promisingtarget for development of new cancer therapies. However, progress indevising strategies for nullifying Survivin has been hampered by a lackof knowledge about its biochemical mechanism of action.

HBXIP was originally isolated as a human protein which binds the viraloncogenic protein, HBX, of the Hepatitis B Virus (HBV) (Melegari, M. etal. 1998 J Virol 72:1737-1743). HBXIP encodes a protein of 9.6-kDa witha putative leucine zipper motif. Expression of HBXIP mRNA has beendemonstrated in essentially all tissues examined to date, and is notlimited to the liver (Melegari, M. et al. 1998 J Virol 72:1737-1743). Inthe context of HBV-infection, HBXIP reportedly reduces viral replicationand abolishes the transactivation function of viral HBX protein(Melegari, M. et al. 1998 J Virol 72:1737-1743), however, little isknown about the physiological roles of HBXIP in human cells.

Thus, there is a need for investigation into the anti-apoptoticmechanism of Survivin and its role in neoplastic diseases as a means fordeveloping novel cancer treatments.

SUMMARY OF THE INVENTION

In one embodiment, the present invention includes a method forregulating cellular apoptosis by affecting interaction of hepatitis BX-interacting protein (HBXIP) with Survivin. A method for enhancingapoptosis of neoplastic cells is described, wherein interaction ofhepatitis B X-interacting protein (HBXIP) with Survivin is inhibited.SiRNA or antisense can be used to downregulate expression of HBXIP. Thelevel of HBXIP can also be reduced in the presence of HBXIP- orSurvivin-specific antibodies. In yet another embodiment, the interactionof HBXIP with Survivin may be inhibited with the use of specificinhibitors, molecular decoys, or the like.

Another aspect of the invention includes a pharmaceutical compositioncomprising a compound that inhibits HBXIP in the presence of Survivin.Yet another embodiment is a method of treating neoplastic diseasecomprising administration of an inhibitor of HBXIP in the presence ofSurvivin. A method for treating human liver disease associated with HBVis also described, comprising administration of an inhibitor of HBXIP inthe presence of Survivin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(a-f) shows differences in Caspase suppression by Survivin andXIAP. Effects of purified Survivin (FIGS. 1 a, 1 c, 1 e) and XIAP (FIGS.1 b, 1 d, 1 f) on recombinant active Caspase-3 (FIGS. 1 a, 1 b) or oncaspase-3-like protease activity induced in cell extracts by Cytochromec (FIGS. 1 c, 1 d, 1 e, 1 f) are shown. In FIGS. 1 a and 1 b, 100 μMrecombinant active Caspase-3 was incubated with recombinant Survivin(FIG. 1 a) or XIAP (FIG. 1 b) or with control protein. In FIGS. 1 c and1 d, purified Survivin (FIG. 1 c) or XIAP (FIG. 1 d), or control proteinwas added concurrently with the addition of Cytochrome C and dATP. InFIGS. 1 e and 1 f, Survivin (FIG. 1 e) or XIAP (FIG. 1 f) was addedafter stimulation with Cytochrome c and dATP. Caspase activity wascontinuously measured, and results are shown in RFU, relativefluorescence units.

FIG. 2(a-h) illustrates that HBXIP directly binds Survivin. FIG. 2 ashows the results of in vitro binding experiments. FIGS. 2 b and 2 cshow gel-sieve chromatography and FIG. 2 d shows Scatchard analysis ofHis₆-HBXIP binding to purified Survivin (untagged). (B, bound Survivin;F, free Survivin). FIG. 2 e shows the results of mapping of region inSurvivin required for binding HBXIP, autoradiography. FIG. 2 f shows theresults of immunoblotting using anti-myc antibody (upper panel),anti-myc (middle panel) or anti-FLAG antibodies (lower panel). FIG. 2 gshows the results of immunoblotting of lysates from untransfected HepG2cells prepared for immunoprecipitation with anti-Survivin antisera withanti-HBXIP or anti-Survivin antibodies. FIG. 2 h shows the results ofimmunoblotting of subcellular fractionation of 293 cells (M, membrane;C, cytoplasmic; N, nuclear) using anti-HBXIP and anti-Survivinantibodies.

FIG. 3(a-e) shows that HBXIP collaborates with Survivin to suppressCaspase-9 activation. FIGS. 3 a-3 c show the effects of purified HBXIPand Survivin on Caspase activity induced by Cytochrome C (FIG. 3 a),Caspase-8 (FIG. 3 b) or Granzyme B (FIG. 3 c), measured as AFC releasefrom Ac-DEVD-AFC in cell extracts. His₆-HBXIP, Survivin (SVV), or thecombination of these proteins was added to 293 cell lysates prior toCytochrome c (and dATP), recombinant active Caspase-8, or Granzyme B.CTR, control proteins tested. FIGS. 3 d and 3 e illustrate the effectsof recombinant HBXIP, Survivin, or the combination of these proteins orvarious control (CTR) proteins on Caspase-9 activity measured by thecleavage of fluorogenic substrate Ac-LEHD-AFC (FIG. 3 d) or proteolyticprocessing of ˜50 kDa pro-Caspase-9 to ˜35 kDa large and ˜12 kDa smallsubunits, analyzed by immunoblotting analysis using anti-Caspase-9antibody (FIG. 3 e). Minus-signs indicate addition of an equivalentamount of a control protein (GST-CD40 or His₆-Traf3) instead of Survivinor His₆-HBXIP.

FIG. 4(a-f) shows that the combination of HBXIP and Survivin inhibitsthe recruitment of pro-Caspase-9 to activated Apaf1. FIG. 4 aillustrates the results of immunoblotting using GST-Survivin, GST-CD40or GST-HBXIP with or without purified Survivin (untagged), incubatedwith His₆-pro-Caspase-9, active His₆-Caspase-9 (lacking CARD domain) orHis₆-pro-Caspase-3. GST-fusion proteins were recovered usingglutathione-Sepharose and bound proteins were detected by immunoblottingusing anti-Caspase-9 or anti-Caspase-3 antisera. An equivalent amount ofproteins was loaded directly in gels as a control (“input”). FIG. 4 bshows the results when ³⁵S-labeled pro-Caspase-9 was incubated withHis₆-Apaf1, Cytochrome C and dATP, in the absence or presence ofGST-HBXIP and Survivin. His₆-Apaf1 and associated proteins wererecovered by adsorption to Ni-resin, and bound proteins were analyzed byautoradiography (for Caspase-9) or immunoblotting using anti-Apaf1 oranti-Cytochrome C antibodies. FIGS. 4 c and 4 d illustrate the resultswhen purified His₆-Apaf1, His₆-pro-Caspase-9, Cytochrome C and dATP wereincubated in the absence (FIG. 4 c) or presence (FIG. 4 d) ofrecombinant purified Survivin and HBXIP. Proteins were analyzed bygel-sieve chromatography, using immunoblotting to detect proteins ineluted fractions. FIG. 4 e shows caspase activity in column fractionsfrom gel-sieve experiments, determined by monitoring the cleavage ofAc-DEVD-AFC after the incubation of each fraction from FIG. 4 c and FIG.4 d with recombinant pro-Caspase-3. FIG. 4 f: (Upper panel) Full-lengthpurified GST-HBXIP and GST-HBXIP (1-40) tested for binding toHis₆-Survivin or control protein (His₆-Traf3). Bound proteins wereanalyzed by immunoblotting using anti-GST antisera (upper panel). Anequivalent amount of GST-fusion protein was loaded directly in gels as acontrol (“input”). (Lower panel) HT1080 cells were transientlytransfected with expression plasmids encoding myc-Survivin, FLAG-HBXIP,FLAG-HBXIP(1-40), Bcl-XL (for Staurosporine [STS]), or CrnA (foranti-Fas), alone or in various combinations, with pEGFP marker plasmid,using pcDNA3 to normalize total DNA content. Then, cells were culturedwith STS or anti-Fas antibody, and the percentage of apoptotic cells wasdetermined by DAPI staining (mean ±SE; n=3) one day later.

FIG. 5(a-e) shows regulation of Caspase activation and apoptosis byendogenous HBXIP. FIG. 5 a shows that HBXIP expression is elevated inHBV-infected liver and hepatocellular cancers. Protein samples from thetumors (T) and non-malignant tissue (NT) of three patients withHBV-related-hepatocellular carcinoma and two normal livers specimens (N1and N2) analyzed by SDS-PAGE/immunoblotting using antisera specific forHBXIP, Survivin or d-Tubulin (as a control). FIG. 5 b illustratescaspase activity in the same tissue samples measured using fluorigenicsubstrate Ac-DEVD-AFC after treatment with Cytochrome c and dATP (leftpanel) or with Granzyme B (right panel). Results are arbitrarilyexpressed relative to Caspase activity generated in normal liverspecimen (N1) (mean ±SE; n=3 determinations). FIG. 5 c illustratesimmunoblotting analysis of HeLa cells transfected with siRNA targetingHBXIP or control RNA (CTR), using anti-HBXIP and anti-α-Tubulin (loadingcontrol) antisera. FIG. 5 d shows caspase activity in HeLa cell extractsafter treatment with HBXIP-siRNA or control-RNA and incubation withCytochrome C and dATP, in the presence or absence recombinant Survivin(left panel) or XIAP (right panel), measured as a release of AFC fromAc-DEVD-AFC substrate (mean ±SE; n=3). FIG. 5 e shows the percentage ofapoptotic cells (mean ±SE; n=3) determined by DAPI-staining followingculture of control-RNA- or HBXIP siRNA-transfected HeLa cells withEtoposide (VP-16) or Staurosporine (STS).

FIG. 6(a-f) shows that HBX associates with Survivin through HBXIP andsuppresses Caspase activation. FIG. 6 a shows immunoblot analysis usinganti-HBX antibody of in vitro protein binding after incubation ofrecombinant His₆-HBX with GST-HBXIP, GST-Survivin, or GST-CD40(control). FIGS. 6 b and 6 c show caspase activity measured in 293 cellextracts in the presence of His₆-HBX or control [CTR] proteins, purifiedHis₆-HBXIP, purified Survivin, or various combinations of these proteinsafter Cytochrome c and dATP were added. FIG. 6 d shows the results ofimmunoprecipitation using anti-Survivin antibody (upper panel) oflysates of HEK 293 cell expressing FLAG-tagged-HBX or FLAG-SIP (as acontrol) together with Myc-Survivin or HA-HBXIP with anti-FLAG epitopeantibody demonstrates increased association of Survivin with HBX whenHBXIP was co-expressed (compare last two lanes at right). FIG. 6 e showsthe results when His₆-pro-Caspase-9 was incubated with GST-HBXIP(+) orGST-CD40 control protein (−), in the presence or absence of His₆-HBX anduntagged Survivin. GST-fusion proteins were recovered onglutathione-Sepharose and bound proteins were detected by immunoblottingusing anti-Caspase-9, anti-Survivin, or anti-HBX antisera. FIG. 6 fshows immunoblotting using anti-Survivin antibody (top panel) of HepG2cell extracts immunodepleted using anti-Survivin antisera or preimmuneserum (CTR). Then extracts were analyzed for Caspase activity based onAc-DEVD-AFC cleavage, where lysates were incubated with recombinant HBX(+) or control protein (−) prior to stimulation with Cytochrome c anddATP.

FIG. 7 shows details of yeast two-hybrid screening using human Survivinas bait.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Survivin is an anti-apoptotic protein of undefined mechanism, which ispathologically over-expressed in most human cancers. It has beendiscovered that Survivin forms complexes with an endogenous cellularprotein, Hepatitis B X-interacting protein (HBXIP), which was originallyrecognized for its association with the X protein of Hepatitis B Virus(HBX). Survivin/HBXIP complexes, but neither Survivin nor HBXIPindividually, bind pro-Caspase-9, preventing its recruitment to Apaf1,and thereby selectively suppress apoptosis initiated via themitochondria/Cytochrome C pathway. Viral HBX protein also interacts withthe Survivin/HBXIP complex and suppresses Caspase activation in aSurvivin-dependent manner. Thus, HBXIP functions as a cofactor forSurvivin, and serves as a link between the cellular apoptosis machineryand a viral pathogen involved on an epidemic scale in hepatocellularcarcinogenesis.

In an effort to provide insights into the anti-apoptotic mechanism ofSurvivin, cDNA libraries were screened for Survivin-binding proteins,resulting in the discovery that hepatitis B X-interacting protein(HBXIP) associates with p17 Survivin. HBXIP was originally identified byvirtue of its ability to interact with the HBX protein of Hepatitis BVirus (HBV) (Melegari, M. et al. 1998 J Virol 72:1737-1743). HBX is aputative oncogenic protein, which has been previously implicated inregulation of apoptosis, as well as other processes (reviewed inMurakami, S. 2001 J Gastroenterol 36:651-660). HBXIP operates as acofactor for Survivin, allowing it to bind and suppress activation ofpro-Caspase-9, the apical protease in a mitochondrial pathway for celldeath. These findings thus provide novel insights into theanti-apoptotic mechanism of Survivin, and provide a link betweenSurvivin and neoplastic diseases associated with HBV infection.

Some embodiments of the present invention relate to methods ofinhibiting the interaction between Survivin and HBXIP both in vitro andin vivo thereby resulting in the enhancement of apoptosis. Methods ofinhibiting such interactions include, but are not limited to, reducingthe level or expression of the Survivin and/or HBXIP protein byinhibiting transcription, stability or processing of the Survivin and/orHBXIP mRNA; inhibiting or altering post-translation processing of theSurvivin and/or HBXIP protein; or interfering with the interactionbetween Survivin and HBXIP.

A number of different methods of inhibiting the interaction betweenSurvivin and HBXIP are disclosed herein. For example, nucleic acids ornucleic acid-like compounds can be used to reduce the expression of theSurvivin and/or HBXIP gene. Such nucleic acids and nucleic acid-likecompounds can include DNA, RNA, peptide nucleic acid (PNA), or modifiedpolynucleotides. Such nucleic acids can function as antisense nucleicacids, catalytic RNAs (ribozymes), and small interfering RNAs (siRNAs),such as dsRNAs. In other embodiments, antibodies can be prepared whichhave affinity for and bind to either Survivin or HBXIP. Such antibodiescan be used to prevent disrupt or otherwise inhibit the interactionbetween Survivin and HBXIP. Molecular decoys can also be used toprevent, disrupt or otherwise inhibit the interaction between Survivinand HBXIP. In some embodiments, the molecular decoys are peptides orpeptidomimetics which comprise the portion of the Survivin or HBXIPpolypeptide which facilitates interaction between Survivin and HBXIP butlacks a sufficient homology with the full-length Survivin or HBXIPpolypeptide to permit formation of a complex that inhibits apoptosis. Insuch embodiments, the peptides or peptidomimetics act as competitiveinhibitors of the interaction between Survivin and HBXIP. In addition tothe above embodiments, small molecules can be used to inhibit theinteraction between Survivin and HBXIP. Such molecules can be obtained,for example, by screening synthetic or natural product libraries.

Other embodiments of the present invention relate to methods ofenhancing the interaction between Survivin and HBXIP both in vitro andin vivo thereby resulting in the inhibition of apoptosis. Methods ofenhancing such interactions include, but are not limited to, increasingthe level or expression of the Survivin and/or HBXIP protein byenhancing transcription and/or stability of the Survivin and/or HBXIPmRNA or facilitating the interaction between Survivin and HBXIP.

A number of different methods of enhancing the interaction betweenSurvivin and HBXIP are disclosed herein. For example, antibodies can beprepared which have affinity for, and thus bind to, an epitope that iscreated upon the formation of the complex that results from the bindingof Survivin and HBXIP. Such antibodies can be used to facilitate complexformation between Survivin and HBXIP. In addition to the aboveembodiments, small molecules can be used to facilitate the interactionbetween Survivin and HBXIP. Such molecules can be obtained, for example,by screening synthetic or natural product libraries. Additionally,methods of increasing the production of a protein by increasing itstranscription and/or translation efficiency can be used to increase theamount of one or both of Survivin and HBXIP thereby increasing theamount of protein available for complex formation.

In some embodiments of the present invention, the cells in whichapoptosis is inhibited or enhanced include, but are not limited to,human cells as well as those of other vertebrate animals including fish,avian, cattle, goat, pig, sheep, rodent, hamster, mouse, rat, andprimate. Cells which are targeted can also be from the germ line orsomatic, totipotent or pluripotent, dividing or non-dividing, parenchymaor epithelium, immortalized or transformed, or the like. The cell may bea stem cell or a differentiated cell. Cell types that are differentiatedinclude adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium,neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages,neutrophils, eosinophils, basophils, mast cells, leukocytes,granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts,hepatocytes, and cells of the endocrine or exocrine glands. In someembodiments of the present invention, the cells are neoplastic cellswithin humans and/or other animals.

Antisense Nucleic Acids

Antisense nucleic acids that are used to reduce the amount of Survivinand/or HBXIP polypeptides that is present inside a cell arecomplementary to at least a portion of the coding strand of eitherSurvivin (SEQ ID NO: 1) or HBXIP (SEQ ID NO: 3). Such antisense nucleicacids include antisense polynucleotides complementary to the full-lengthsense strand of Survivin and/or HBXIP or oligonucleotide fragments fromat least about 15 to more than about 120 nucleotides, including at leastabout 16 nucleotides, at least about 17 nucleotides, at least about 18nucleotides, at least about 19 nucleotides, at least about 20nucleotides, at least about 21 nucleotides, at least about 22nucleotides, at least about 23 nucleotides, at least about 24nucleotides, at least about 25 nucleotides, at least about 26nucleotides, at least about 27 nucleotides, at least about 28nucleotides, at least about 29 nucleotides, at least about 30nucleotides, at least about 35 nucleotides, at least about 40nucleotides, at least about 45 nucleotides, at least about 50nucleotides, at least about 55 nucleotides, at least about 60nucleotides, at least about 65 nucleotides, at least about 70nucleotides, at least about 75 nucleotides, at least about 80nucleotides, at least about 85 nucleotides, at least about 90nucleotides, at least about 95 nucleotides, at least about 100nucleotides, at least about 110 nucleotides, at least about 120nucleotides or greater than 120 nucleotides.

As used herein, the term “oligonucleotide” refers to an oligomer orpolymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) ormimetics thereof. This term includes oligonucleotides composed ofnaturally-occurring nucleobases, sugars and covalent internucleoside(backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

While antisense oligonucleotides are a preferred form of antisensecompound, embodiments of the present invention contemplates otheroligomeric antisense compounds, including but not limited to,oligonucleotide mimetics such as are described below. The antisenseoligonucleotides described herein also include ribozymes, external guidesequence (EGS) oligonucleotides (oligozymes), and other short catalyticRNAs or catalytic oligonucleotides which hybridize to the target nucleicacid and modulate its expression.

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn the respective ends of this linear polymericstructure can be further joined to form a circular structure, however,open linear structures are generally preferred. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the internucleoside backbone of the oligonucleotide. Thenormal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiesterlinkage.

Specific examples of antisense compounds useful in certain embodimentsof this invention include oligonucleotides containing modified backbonesor non-natural internucleoside linkages. As used herein,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

In some embodiments modified oligonucleotide backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand borano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Certain oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

In some embodiments modified oligonucleotide backbones that do notinclude a phosphorus atom therein have backbones that are formed byshort chain alkyl or cycloalkyl internucleoside linkages, mixedheteroatom and alkyl or cycloalkyl internucleoside linkages, or one ormore short chain heteroatomic or heterocyclic internucleoside linkages.These include those having morpholino linkages (formed in part from thesugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxideand sulfone backbones; formacetyl and thioformacetyl backbones;methylene formacetyl and thioformacetyl backbones; riboacetyl backbones;alkene containing backbones; sulfamate backbones; methyleneimino andmethylenehydrazino backbones; sulfonate and sulfonamide backbones; amidebackbones; and others having mixed N, O, S and CH₂ component parts.

In other embodiments, oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

In still other embodiments of the present invention, the expression ofSurvivin and/or HBXIP is modulated using oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones. Modified oligonucleotides may also contain one or moresubstituted sugar moieties. In some embodiments oligonucleotidescomprise one of the following at the 2′ position: OH; F; O-, S-, orN-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl,wherein the alkyl, alkenyl and alkynyl may be substituted orunsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl.Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃,O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂ andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl,alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl,Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties.Another modification includes 2′-methoxyethoxy(2′OCH₂CH₂OCH₃, also knownas 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta,1995, 78, 486-504).

An embodiment of the present invention includes the use of LockedNucleic Acids (LNAs) to generate antisense nucleic acids having enhancedaffinity and specificity for the target polynucleotide. LNAs are nucleicacid in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbonatom of the sugar ring thereby forming a bicyclic sugar moiety. Thelinkage is preferably a methelyne (—CH₂-)n group bridging the 2′ oxygenatom and the 4′ carbon atom wherein n is 1 or 2. LNAs and preparationthereof are described in WO 98/39352 and WO 99/14226.

Other modifications include 2′-methoxy(2′-O—CH₃),2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH═CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar.

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine, 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine and otheralkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further modified nucleobases include tricyclicpyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazi-n-2(3H)-one), phenothiazine cytidine(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as asubstituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrimido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modifiednucleobases may also include those in which the purine or pyrimidinebase is replaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the oligomeric compoundsdescribed herein. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Another modification of the antisense oligonucleotides described hereininvolves chemically linking to the oligonucleotide one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. The antisense oligonucleotidescan include conjugate groups covalently bound to functional groups suchas primary or secondary hydroxyl groups. Conjugate groups includeintercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, polyethers, groups that enhance the pharmacodynamic propertiesof oligomers, and groups that enhance the pharmacokinetic properties ofoligomers. Typical conjugates groups include cholesterols, lipids,phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups thatenhance the pharmacodynamic properties, in the context of thisinvention, include groups that improve oligomer uptake, enhance oligomerresistance to degradation, and/or strengthen sequence-specifichybridization with RNA. Groups that enhance the pharmacokineticproperties, in the context of this invention, include groups thatimprove oligomer uptake, distribution, metabolism or excretion.Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-5-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., dihexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylaminocarbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The methods describedherein also contemplate the use of antisense compounds which arechimeric compounds. “Chimeric” antisense compounds or “chimeras,” asused herein, are antisense compounds, particularly oligonucleotides,which contain two or more chemically distinct regions, each made up ofat least one monomer unit, i.e., a nucleotide in the case of anoligonucleotide compound. These oligonucleotides typically contain atleast one region wherein the oligonucleotide is modified so as to conferupon the oligonucleotide increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the oligonucleotide mayserve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, RNase H is a cellular endonuclease whichcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of oligonucleotide inhibition of geneexpression. Consequently, comparable results can often be obtained withshorter oligonucleotides when chimeric oligonucleotides are used,compared to phosphorothioate deoxyoligonucleotides hybridizing to thesame target region. Cleavage of the RNA target can be routinely detectedby gel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art.

Chimeric antisense compounds for use in the methods of the presentinvention may be formed as composite structures of two or moreoligonucleotides, modified oligonucleotides, oligonucleosides and/oroligonucleotide mimetics as described above. Such compounds have alsobeen referred to in the art as hybrids or gapmers.

The antisense compounds used in accordance with some embodiments of thisinvention may be conveniently and routinely made through the well-knowntechnique of solid phase synthesis. Equipment for such synthesis is soldby several vendors including, for example, Applied Biosystems (FosterCity, Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

The antisense compounds for use with the methods described hereinencompass any pharmaceutically acceptable salts, esters, or salts ofsuch esters, or any other compound which, upon administration to ananimal including a human, is capable of providing (directly orindirectly) the biologically active metabolite or residue thereof.

In some embodiments of the present invention, an antisense nucleic acidspecific to the Survivin and/or the HBXIP gene is synthesized andintroduced directly into a subject. In other embodiments, the antisensenucleic acid can be formulated as part of a targeted delivery system,such as a target specific liposome, which specifically recognizes anddelivers the antisense nucleic acid to an appropriate tissue or celltype, such as a tumor cell. Upon administration of the targetedantisense nucleic acid to a subject, the antisense nucleic acid isdelivered to the appropriate cell type thereby increasing theconcentration antisense nucleic acid within the cell type.

In other embodiments of the present invention, an appropriate cell ortissue is provided with expression construct which comprises a nucleicacid that encodes the antisense RNA that is specific to the Survivinand/or the HBXIP gene. In these embodiments, the nucleic acid thatencoding the antisense RNA can be placed under the control of either aconstitutive or a regulatable promoter.

Interfering RNA

Some embodiments of the present invention provide a method of producingsequence-specific inhibition of the expression of either the Survivinand/or the HBXIP gene by introducing a small inhibitory RNA (siRNA). Asused herein siRNAs are synonymous with double-stranded RNA (dsRNA), andinclude double-stranded RNA oligomers with or without hairpin structuresat each end. Small interfering RNAs comprise oligonucleotides of atleast about 15 to greater than about 120 nucleotides, including at leastabout 16 nucleotides, at least about 17 nucleotides, at least about 18nucleotides, at least about 19 nucleotides, at least about 20nucleotides, at least about 21 nucleotides, at least about 22nucleotides, at least about 23 nucleotides, at least about 24nucleotides, at least about 25 nucleotides, at least about 26nucleotides, at least about 27 nucleotides, at least about 28nucleotides, at least about 29 nucleotides, at least about 30nucleotides, at least about 35 nucleotides, at least about 40nucleotides, at least about 45 nucleotides, at least about 50nucleotides, at least about 55 nucleotides, at least about 60nucleotides, at least about 65 nucleotides, at least about 70nucleotides, at least about 75 nucleotides, at least about 80nucleotides, at least about 85 nucleotides, at least about 90nucleotides, at least about 95 nucleotides, at least about 100nucleotides, at least about 110 nucleotides, at least about 120nucleotides or greater than 120 nucleotides. In certain embodiments ofthe present invention, the siRNA comprises an oligonucleotide from about21 to about 25 nucleotides in length. In some embodiments, the siRNAmolecule is a heteroduplex of RNA and DNA.

As with antisense nucleic acids, siRNAs can include modifications toeither the phosphate-sugar backbone or the nucleoside. For example, thephosphodiester linkages of natural RNA may be modified to include atleast one of a nitrogen or sulfur heteroatom. Modifications in RNAstructure may be tailored as described for antisense nucleic acids.

A process for inhibiting expression of the Survivin and/or the HBXIPgene in a cell comprises introduction of an siRNA with partial or fullydouble-stranded character into the cell. Inhibition is sequence-specificin that a nucleotide sequence from a portion of the Survivin and/or theHBXIP gene is chosen to produce inhibitory RNA. Depending on the dose ofsiRNA delivered, this process can provide partial or complete loss offunction for the Survivin and/or the HBXIP gene.

In some embodiments of the present invention, an siRNA specific to theSurvivin and/or the HBXIP gene is synthesized and introduced directlyinto a subject. In other embodiments, the siRNA can be formulated aspart of a targeted delivery system, such as a target specific liposome,which specifically recognizes and delivers the siRNA to an appropriatetissue or cell type, such as a tumor cell. Upon administration of thetargeted siRNA to a subject, the siRNA is delivered to the appropriatecell type, thereby increasing the concentration siRNA within the celltype.

In other embodiments of the present invention, an appropriate cell ortissue is provided with expression construct which comprises a nucleicacid that encodes one or both strands of an siRNA that is specific tothe Survivin and/or the HBXIP gene. In these embodiments, the nucleicacid that encodes one or both strands of the siRNA can be placed underthe control of either a constitutive or a regulatable promoter. In someembodiments, the nucleic acid encodes an siRNA that forms a hairpinstructure.

Inhibition of Gene Expression Using Nucleic Acids

Inhibition of gene expression refers to the absence (or observabledecrease) in the level of protein and/or mRNA product from the Survivinand/or the HBXIP gene. The consequences of inhibition can be confirmedby examination of the outward properties of the cell or organism, suchas increased apoptosis, or by biochemical techniques, such asdetermining the amount of Caspase-9 activity or directly measuringlevels of the Survivin and/or the HBXIP transcript. For a cell line orwhole organism, gene expression is conveniently assayed by use of areporter or drug resistance gene whose protein product is easilyassayed. Such reporter genes include acetohydroxyacid synthase (AHAS),alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase(GUS), chloramphenicol acetyltransferase (CAT), green fluorescentprotein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopalinesynthase (NOS), octopine synthase (OCS), and derivatives thereof.Multiple selectable markers are available that confer resistance toampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin,kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, andtetracyclin.

Depending on the assay, quantitation of the amount of gene expressionallows one to determine a degree of inhibition which is greater than10%, 33%, 50%, 90%, 95% or 99% as compared to an untreated cell. Lowerdoses of injected material and longer times after administration of theantisense nucleic acid, catalytic RNA or siRNA may result in inhibitionin partial inhibition of the Survivin and/or HBXIP genes.

Antisense nucleic acids, catalytic RNAs and siRNAs comprising anucleotide sequences identical to a portion of the Survivin and/or HBXIPgenes are contemplated in some embodiments of the present invention.However, nucleic acid sequences with insertions, deletions, and singlepoint mutations relative to the target sequence are also effective forinhibition of gene expression. Thus, sequence identity may optimized bysequence comparison and alignment algorithms known in the art (seeGribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991,and references cited therein) and calculating the percent differencebetween the nucleotide sequences by, for example, the Smith-Watermanalgorithm as implemented in the BESTFIT software program using defaultparameters (e.g., University of Wisconsin Genetic Computing Group).Greater than 90% sequence identity, or even 100% sequence identity,between the siRNA and the portion of the target gene is preferred.Alternatively, the duplex region of the RNA may be defined functionallyas a nucleotide sequence that is capable of hybridizing with a portionof the target gene transcript. Exemplary hybridization conditions are400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C.hybridization for 12-16 hours; followed by washing.

Antibodies

Some embodiments of the present invention related to the use ofantibodies that bind to an epitope present on the Survivin polypeptide(SEQ ID NO: 2) or the HBXIP polypeptide (SEQ ID NO: 4) to interfere withthe interaction between Survivin and HBXIP thereby resulting in anenhancement of apoptosis. Other embodiments of the present inventionrelate to the use of antibodies which recognize an epitope that isspecific to the formed Survivin/HBXIP complex thereby enhancing theinteraction between Survivin and HBXIP. Enhancement of Survivin/HBXIPinteraction results in an inhibition of apoptosis.

Antibodies and fragments can be made by standard methods (See, forexample, E. Harlow et al., Antibodies, A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1988). However, theisolation, identification, and molecular construction of antibodies hasbeen developed to such an extent that the choices are almostinexhaustible. Therefore, examples of antibody parts, and complexes willbe provided with the understanding that this can only represent asampling of what is available.

In one embodiment of the present invention, the antibody is a singlechain Fv region. Antibody molecules have two generally recognizedregions, in each of the heavy and light chains. These regions are theso-called “variable” region which is responsible for binding to thespecific antigen in question, and the so-called “constant” region whichis responsible for biological effector responses such as complementbinding, binding to neutrophils and macrophages, etc. The constantregions are not necessary for antigen binding. Accordingly, constantregions can be separated from the antibody molecule leaving only thevariable binding regions.

The variable regions of an antibody are composed of a light chain and aheavy chain. Light and heavy chain variable regions have been cloned andexpressed in foreign hosts, while maintaining their binding ability.Therefore, it is possible to generate a single chain structure from themultiple chain aggregate (the antibody), such that the single chainstructure will retain the three-dimensional architecture of the multiplechain aggregate.

Fv fragments which are single polypeptide chain binding proteins havingthe characteristic binding ability of multi-chain variable regions ofantibody molecules, can be used in the methods described herein. Thesefragments are produced, for example, following the methods of Ladner etal., U.S. Pat. No. 5,260,203, issued Nov. 9, 1993, using a computerbased system and method to determine chemical structures. These chemicalstructures are used for converting two naturally aggregated butchemically separated light and heavy polypeptide chains from an antibodyvariable region into a single polypeptide chain which will fold into athree dimensional structure very similar to the original structure ofthe two polypeptide chains. The two regions may be linked using an aminoacid sequence as a bridge.

The single polypeptide chain obtained from this method can then be usedto prepare a genetic sequence coding therefor. The genetic sequence canthen be replicated in appropriate hosts, further linked to controlregions, and transformed into expression hosts, wherein it can beexpressed. The resulting single polypeptide chain binding protein, uponrefolding, has the binding characteristics of the aggregate of theoriginal two (heavy and light) polypeptide chains of the variable regionof the antibody.

In a further embodiment, the antibodies are multivalent forms ofsingle-chain antigen-binding proteins. Multivalent forms of single-chainantigen-binding proteins have significant utility beyond that of themonovalent single-chain antigen-binding proteins. A multivalentantigen-binding protein has more than one antigen-binding site whichresults in an enhanced binding affinity. The multivalent antibodies canbe produced using the method disclosed in Whitlow et al., U.S. Pat. No.5,869,620, issued Feb. 9, 1999. The method involves producing amultivalent antigen-binding protein by linking at least two single-chainmolecules, each single chain molecule having two binding portions of thevariable region of an antibody heavy or light chain linked into a singlechain protein. In this way the antibodies can have binding sites fordifferent parts of an antigen or have binding sites for multipleantigens.

In one embodiment, the antibody is an oligomer. The oligomer is producedas in PCT/EP97/05897, filed Oct. 24, 1997, by first isolating a specificligand from a phage-displayed library. Oligomers overcome the problem ofthe isolation of mostly low affinity ligands from these libraries, byoligomerizing the low-affinity ligands to produce high affinityoligomers. The oligomers are constructed by producing a fusion proteinwith the ligand fused to a semi-rigid hinge and a coiled coil domainfrom Cartilage Oligomeric Matrix Protein (COMP). When the fusion proteinis expressed in a host cell, it self assembles into oligomers.

In some embodiments, the oligomers are peptabodies (Terskikh et al.,Biochemistry 94:1663-1668 (1997)). Peptabodies can be exemplified as IgMantibodies which are pentameric with each binding site havinglow-affinity binding, but able to bind in a high affinity manner as acomplex. Peptabodies are made using phage-displayed random peptidelibraries. A short peptide ligand from the library is fused via asemi-rigid hinge at the N-terminus of the COMP (cartilage oligomericmatrix protein) pentamerization domain. The fusion protein is expressedin bacteria where it assembles into a pentameric antibody which showshigh affinity for its target. Depending on the affinity of the ligand,an antibody with very high affinity can be produced.

In some embodiments the antibody, antibody part or antibody complex isderived from humans or is “humanized” (i.e. non-immunogenic in a human)by recombinant or other technology. Such antibodies are the equivalentsof the monoclonal and polygonal antibodies disclosed herein, but areless immunogenic, and are better tolerated by the patient.

Humanized antibodies may be produced, for example, by replacing animmunogenic portion of an antibody with a corresponding, butnon-immunogenic portion (i.e. chimeric antibodies) (See, for example,Robinson, et al., PCT Application No. PCT/US86/02269; Akira, et al.,European Patent Application No. 184,187; Taniguchi, European PatentApplication No. 171,496; Morrison, et al., European Patent ApplicationNo. 173,494; Neuberger, et al., International Patent Publication No.WO86/01533; Cabilly, et al., European Patent Application No. 125,023;Better, et al., Science 240:1041-1043 (1988); Liun, et al., Proc. Natl.Acad. Sci. USA 84:3439-3433 (1987); Liu, et al., J. Immunol.139:3521-3526 (1987); Sun, et al., Proc. Natl. Acad. Sci. USA 84:214-218(1987); Nishimura, et al., Canc. Res. 47:999-1005 (1987); Wood, et al.,Nature 314:446-449 (1985)); Shaw et al., J. Natl. Cancer Inst.80:1553-1559 (1988); all of which references are incorporated herein byreference). General reviews of “humanized” chimeric antibodies areprovided by Morrison, (Science, 229:1202-1207 (1985)) and by Oi, et al.,BioTechniques 4:214 (1986); the disclosures of which are incorporatedherein by reference in their entireties).

Suitable “humanized” antibodies can be alternatively produced by CDR orCEA substitution (Jones, et al., Nature 321:552-525 (1986); Verhoeyan etal., Science 239:1534 (1988); Bsidler, et al., J. Immunol. 141:4053-4060(1988); the disclosures of which are incorporated herein by reference intheir entireties.

Small Molecules

Screening Chemical Libraries

Having identified the interaction between Survivin and HBXIP as involvedin the inhibition of apoptosis, the present invention furthercontemplates the use of these expressed target proteins in assays toscreen libraries of compounds for candidates molecules that inhibit theinteraction between Survivin and HBXIP. The generation of chemicallibraries is well known in the art. A combinatorial chemical library isa collection of diverse chemical compounds generated by either chemicalsynthesis or biological synthesis by combining a number of chemical“building block” reagents. For example, a linear combinatorial chemicallibrary such as a polypeptide library is formed by combining amino acidsin every possible combination to yield peptides of a given length.Millions of chemical compounds theoretically can be synthesized throughsuch combinatorial mixings of chemical building blocks. For example, onecommentator observed that the systematic, combinatorial mixing of 100interchangeable chemical building blocks results in the theoreticalsynthesis of 100 million tetrameric compounds or 10 billion pentamericcompounds. (Gallop et al., Journal of Medicinal Chemistry, Vol. 37, No.9, 1233-1250 (1994). Other chemical libraries known to those in the artmay also be used, including natural product libraries.

Once generated, combinatorial libraries can be screened for compoundsthat possess desirable biological properties. For example, the abilityto enhance apoptosis in cancer cells would be a useful property of acompound that can function as or be developed into an anticancer drug.One mechanism by which the compound can enhance apoptosis is through theinhibition of the interaction between Survivin and HBXIP.

To illustrate the screening process, Survivin, HBXIP and chemicalcompounds of the library are combined and permitted to interact with oneanother in the presence of pro-Caspase-9. The activation ofpro-Caspase-9 is measured in the presence of Survivin, HBXIP and thecandidate chemical compound. This activation is compared with theactivation of pro-Caspase-9 that is measured in the presence Survivinand HBXIP without the candidate chemical compound. If the candidatecompound inhibits the interaction between Survivin and HBXIP, the amountof activation of pro-Caspase-9 will increase compared to the amountpro-Caspase-9 activation in the presence of Survivin and HBXIP withoutcandidate compound. The characteristics of each library compound areencoded so that compounds demonstrating activity that prevents orotherwise disrupts the interaction between Survivin and HBXIP can beanalyzed and features common to the various compounds identified can beisolated and combined into future iterations of libraries.

Once a library of compounds is screened, subsequent libraries aregenerated using those chemical building blocks that possess the featuresshown in the first round of screening to have activity that prevents orotherwise disrupts the interaction between Survivin and HBXIP. Usingthis method, subsequent iterations of candidate compounds will possessmore and more of those structural and functional features necessary toprevent or otherwise disrupt the interaction between Survivin and HBXIP,until a group of compounds with the ability to substantially inhibit theinteraction between Survivin and HBXIP.

It will be appreciated that in addition to small molecule libraries,libraries of other compounds, such as antibodies, peptides, nucleicacids and other molecules described herein, can be used to identifycompounds which inhibit the interaction between Survivin and HBXIP.

Pharmaceutical Compositions

Some embodiments of the present invention also include pharmaceuticalcompositions and formulations which comprise the therapeutic compoundsdescribed herein (that is, the antisense nucleic acids, catalytic RNAs,siRNAs, antibodies, peptides, peptidomimetics, small molecules and othercompounds which inhibit or enhance the interaction of Survivin andHBXIP). Such pharmaceutical compositions may be administered in a numberof ways depending upon whether local or systemic treatment is desiredand upon the area to be treated. Administration may be topical(including ophthalmic and to mucous membranes including vaginal andrectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be desirable. In some embodiments, topical formulationsinclude those in which the therapeutic compounds described herein are inadmixture with a topical delivery agent such as lipids, liposomes, fattyacids, fatty acid esters, steroids, chelating agents and surfactants.Exemplary lipids and liposomes include neutral (e.g.dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl cholineDMPC, distearolyphosphatidyl choline) negative (e.g.dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). Therapeutic compounds described herein may beencapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, the therapeuticcompounds described herein can be complexed to lipids, in particular tocationic lipids. Preferred fatty acids and esters include but are notlimited arachidonic acid, oleic acid, eicosanoic acid, lauric acid,caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid,linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein,dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, anacylcarnitine, an acylcholine, or a C₁₋₁₀ alkyl ester (for example,isopropylmyristate, IPM), monoglyceride, diglyceride or pharmaceuticallyacceptable salt thereof.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. In some embodiments, oralformulations are those in which the therapeutic compounds describedherein are administered in conjunction with one or more penetrationenhancers surfactants and chelators. In certain embodiments, surfactantsinclude fatty acids and/or esters or salts thereof, bile acids and/orsalts thereof. Exemplary bile acids/salts include chenodeoxycholic acid(CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid,dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid,glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate. Exemplaryfatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,I-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (for example, sodium). Some embodiments include combinations ofpenetration enhancers, for example, fatty acids/salts in combinationwith bile acids/salts. Another exemplary combination is the sodium saltof lauric acid, capric acid and UDCA. Further penetration enhancersinclude polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cety-1 ether.The therapeutic compounds described herein can be delivered orally ingranular form including sprayed dried particles, or complexed to formmicro or nanoparticles. Complexing agents include poly-amino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Other exemplary complexing agents includechitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine,polyornithine, polyspermines, protamine, polyvinylpyridine,polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g.p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG).

Compositions and formulations for parenteral, intrathecal orintraventricular administration of the therapeutic compounds describedherein can include sterile aqueous solutions which may also containbuffers, diluents and other suitable additives such as, but not limitedto, penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

In some embodiments of the present invention, pharmaceuticalcompositions include, but are not limited to, solutions, emulsions, andliposome-containing formulations. These compositions may be generatedfrom a variety of components that include, but are not limited to,preformed liquids, self-emulsifying solids and self-emulsifyingsemisolids.

In some embodiments of the present invention, pharmaceuticalformulations, which may conveniently be presented in unit dosage form,may be prepared according to conventional techniques well known in thepharmaceutical industry. Such techniques include the step of bringinginto association the active ingredients with the pharmaceuticalcarrier(s) or excipient(s). In general the formulations are prepared byuniformly and intimately bringing into association the activeingredients with liquid carriers or finely divided solid carriers orboth, and then, if necessary, shaping the product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

Emulsions

In some embodiments of the present invention, pharmaceuticalcompositions may be prepared and formulated as emulsions. Emulsions aretypically heterogenous systems of one liquid dispersed in another in theform of droplets usually exceeding 0.1 μm in diameter. (Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al.,in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa., 1985, p. 301). Emulsions are often biphasic systems comprising oftwo immiscible liquid phases intimately mixed and dispersed with eachother. In general, emulsions may be either water-in-oil (w/o) or of theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions may contain additional componentsin addition to the dispersed phases and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199). Emulsion formulations for oral delivery have beenvery widely used because of reasons of ease of formulation, efficacyfrom an absorption and bioavailability standpoint. (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

In one embodiment of the present invention, a pharmaceutical compositioncomprising a therapeutic compound described herein is formulated as amicroemulsion. A microemulsion may be defined as a system of water, oiland amphiphile which is a single optically isotropic andthermodynamically stable liquid solution (Rosoff, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions aresystems that are prepared by first dispersing an oil in an aqueoussurfactant solution and then adding a sufficient amount of a fourthcomponent, generally an intermediate chain-length alcohol to form atransparent system. Therefore, microemulsions have also been describedas thermodynamically stable, isotropically clear dispersions of twoimmiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215). Microemulsions commonly areprepared via a combination of three to five components that include oil,water, surfactant, cosurfactant and electrolyte. Whether themicroemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) typeis dependent on the properties of the oil and surfactant used and on thestructure and geometric packing of the polar heads and hydrocarbon tailsof the surfactant molecules (Schott, in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously. Surfactants used in thepreparation of microemulsions include, but are not limited to, ionicsurfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleylethers, polyglycerol fatty acid esters, tetraglycerol monolaurate(ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate(PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate(MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate(SO750), decaglycerol decaoleate (DAO750), alone or in combination withcosurfactants. The cosurfactant, usually a short-chain alcohol such asethanol, 1-propanol, and 1-butanol, serves to increase the interfacialfluidity by penetrating into the surfactant film and consequentlycreating a disordered film because of the void space generated amongsurfactant molecules. Microemulsions may, however, be prepared withoutthe use of cosurfactants and alcohol-free self-emulsifying microemulsionsystems are known in the art. The aqueous phase may typically be, but isnot limited to, water, an aqueous solution of the drug, glycerol,PEG300, PEG400, polyglycerols, propylene glycols, and derivatives ofethylene glycol. The oil phase may include, but is not limited to,materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters,medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylatedglyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides,saturated polyglycolized C8-C10 glycerides, vegetable oils and siliconeoil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

In some embodiments of the present invention, microemulsions can alsocontain additional components and additives such as sorbitanmonostearate (Grill 3), Labrasol, and penetration enhancers to improvethe properties of the formulation and to enhance the absorption of thetherapeutic compounds described herein. Penetration enhancers used inthe microemulsions can be classified as belonging to one of five broadcategories—surfactants, fatty acids, bile salts, chelating agents, andnon-chelating non-surfactants (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 92). Each of these classeshas been discussed above.

Liposomes

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used herein, the term “liposome” means a vesicle composedof amphiphilic lipids arranged in a spherical bilayer or bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include, but are not limited to, thefollowing: (1) liposomes obtained from natural phospholipids arebiocompatible and biodegradable; (2) liposomes can incorporate a widerange of water and lipid soluble drugs; and (3) liposomes can protectencapsulated drugs in their internal compartments from metabolism anddegradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 245). Important considerations in the preparation of liposomeformulations are the lipid surface charge, vesicle size and the aqueousvolume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes. As the mergingof the liposome and cell progresses, the liposomal contents are emptiedinto the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver intothe skin agents ranging from the size of small molecules tohigh-molecular weight DNA. Compounds including analgesics, antibodies,hormones and high-molecular weight DNAs have been administered to theskin. The majority of applications result in the targeting of the upperepidermis.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap moleculesrather than complex with them. pH-sensitive liposomes have been used todeliver various types of molecules to cells in experimental animals andcells in culture.

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g. as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/po-lyoxyethylene-10-stearyl ether) and Novasome™II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether)were used to deliver cyclosporin-A into the dermis of mouse skin.Results indicated that such non-ionic liposomal systems were effectivein facilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S. T. P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside GMI, galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphat-idylcholine are disclosed in WO 97/13499 (Limet al.).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C.sub.1215G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blurne et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. PP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.)describe PEG-containing liposomes that can be further derivatized withfunctional moieties on their surfaces.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of the therapeutic compoundsdescribed herein, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs may cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the abovementioned classes of penetration enhancers are described below ingreater detail.

Surfactants: In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of oligonucleotides through the mucosais enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether.

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C1-10 alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (for example, oleate,laurate, caprate, myristate, palmitate, stearate, and linoleate).

Bile salts: The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins. Variousnatural bile salts, and their synthetic derivatives, act as penetrationenhancers. Thus the term “bile salts” includes any of the naturallyoccurring components of bile as well as any of their syntheticderivatives. Exemplary bile salts include, for example, cholic acid (orits pharmaceutically acceptable sodium salt, sodium cholate),dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodiumdeoxycholate), glucholic acid (sodium glucholate), glycholic acid(sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate),taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodiumtaurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate),ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate(STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether(POE).

Chelating Agents: Chelating agents, as used in connection with thetherapeutic compounds described herein, can be defined as compounds thatremove metallic ions from solution by forming complexes therewith. Forexample, the chelation of metal ions enhances the absorption ofoligonucleotides through mucosa. In addition to the use of chelatingagents as penetration enhancers, with respect to the nucleic acidcompounds described herein, chelating agents have the added advantage ofalso serving as nuclease inhibitors. Exemplary chelating agents include,but are not limited to, disodium ethylenediaminetetraacetate (EDTA),citric acid, salicylates (for example, sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines).

Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption of the compoundsdescribed herein through the alimentary mucosa. This class ofpenetration enhancers include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives; and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone.

Agents that enhance uptake of oligonucleotides at the cellular level mayalso be added to the pharmaceutical and other compositions of thepresent invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof oligonucleotides.

Other agents may be utilized to enhance the penetration of thetherapeutic compounds described herein, including glycols such asethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones,and terpenes such as limonene and menthone.

Excipients

As used herein, a “pharmaceutical carrier” or “excipient” is apharmaceutically acceptable solvent, suspending agent or any otherpharmacologically inert vehicle for delivering one or more of thetherapeutic compounds described herein. The excipient may be liquid orsolid and is selected, with the planned manner of administration inmind, so as to provide for the desired bulk, consistency, etc., whencombined with a therapeutic compounds described herein and the othercomponents of a given pharmaceutical composition. Typical pharmaceuticalcarriers include, but are not limited to, binding agents (e.g.,pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose, etc.); fillers (e.g., lactose and other sugars,microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates or calcium hydrogen phosphate, etc.);lubricants (e.g., magnesium stearate, talc, silica, colloidal silicondioxide, stearic acid, metallic stearates, hydrogenated vegetable oils,corn starch, polyethylene glycols, sodium benzoate, sodium acetate,etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); andwetting agents (e.g., sodium lauryl sulphate, etc.).

Pharmaceutically acceptable organic or inorganic excipient suitable fornon-parenteral administration which do not deleteriously react withtherapeutic compounds described herein can also be used to formulate thepharmaceutical compositions of the present invention. Suitablepharmaceutically acceptable carriers include, but are not limited to,water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose,amylose, magnesium stearate, talc, silicic acid, viscous paraffin,hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Formulations for topical administration of therapeutic compoundsdescribed herein may include sterile and non-sterile aqueous solutions,non-aqueous solutions in common solvents such as alcohols, or solutionsof the nucleic acids in liquid or solid oil bases. The solutions mayalso contain buffers, diluents and other suitable additives.Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can be used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Dosing

Formulation of the therapeutic compounds described herein and theirsubsequent administration is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonucleotides,and can generally be estimated based on EC₅₀ found to be effective in invitro and in vivo animal models. In general, dosage is from 0.01 μg to100 g per kg of body weight, and may be given once or more daily,weekly, monthly or yearly, or even once every 2 to 20 years. Persons ofordinary skill in the art can easily estimate repetition rates fordosing based on measured residence times and concentrations of the drugin bodily fluids or tissues. Following successful treatment, it may bedesirable to have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 μg to 100 g per kgof body weight, once or more daily, to once every 20 years.

Structure Activity Relationships

In some embodiments of the present invention, sites of interactionbetween Survivin and HBXIP are identified. Such sites can be a site onSurvivin to which HBXIP binds or a site on HBXIP to which Survivinbinds. These binding sites can be used as potential targets forcompounds that interfere with or otherwise disrupt the interactionbetween Survivin and HBXIP. Sites of interaction between Survivin andHBXIP can be determined using structural biology methods including, butnot limited to, nuclear magnetic resonance spectroscopy, x-raydiffraction, computer-based structural predication methods andcomputer-based molecular threading.

In certain embodiments of the present invention, transverserelaxation-optimized spectroscopy (TROSY) is used to determine site ofinteraction between Survivin and HBXIP. For example, TROSY can be usedto determine binding sites for HBXIP that are present on the Survivinprotein or alternatively binding sites for Survivin that are present onHBXIP. Once the three-dimensional structure of such binding sites areidentified, candidate compounds that potentially have affinity for oneor more of these sites can be identified then tested to determine theirability to interact with these binding sites. In addition to TROSY,other spectroscopic methods, such as cross relaxation-enhancedpolarization transfer NMR (CRINEPT) can be used to determine sites ofinteraction between Survivin and HBXIP. The techniques of CRINEPT andTROSY have been described elsewhere such as in U.S. Pat. No. 6,396,267,the disclosure of which is incorporated herein by reference in itsentirety.

In some embodiments of the present invention, methods for screeningcompounds using spectral techniques to determine binding of compounds tothe HBXIP binding site of Survivin or to the Survivin binding site ofHBXIP are contemplated. Such methods identify compounds that may beuseful in altering the association of HBXIP and Survivin. Compounds maybe screened by using a combination of Nuclear Magnetic Resonance (NMR)binding assays, Fluorescence Polarization Assay (FPA) andComputational-Docking studies.

Some embodiments of this invention are further illustrated by thefollowing examples which should not be construed as limiting. It will beappreciated by those of skill in the art that the techniques disclosedin the examples which follow represent techniques discovered by theinventor to function well in the practice of the embodiments of theinvention described herein, and thus can be considered to constitutepreferred modes for the practice of these embodiments. Those of skill inthe art will, however, in light of the present disclosure, appreciatethat many changes can be made in the specific embodiments which aredisclosed herein and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

EXAMPLE 1 Survivin Differs from XIAP in Caspase Inhibitory Activity

Over-expression of various IAP-family proteins can suppress Caspaseactivation and apoptosis induced by various stimuli. The effects ofSurvivin were compared with XIAP, an extensively studied IAP-familymember which has been shown unequivocally to directly bind and suppresscertain Caspases, including Caspases-3, 7, and 9 (Deveraux, Q. L. &Reed, J. C. 1999 Genes Dev 13:239-252; Deveraux, Q. L. et al. 1997Nature 388:300-304; Sun, C. et al. 1999 Nature 401:818-821; Riedl, S. J.et al. 2001 Cell 104:791-800).

Primary hepatocellular carcinoma tissues and their correspondingnon-cancerous regions were obtained from patients undergoing biopsy orsurgery at Kyoto University Hospital after obtaining informed consent.Cytosolic extracts from cells or liver tissues were prepared in buffer A(10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1mM PMSF and 20 mM HEPES-KOH pH 7.4), as described previously (Deveraux,Q. L. et al 1997 Nature 388:300-304). After measuring protein levels(Biorad, Hercules, Calif.), 20 μg of protein sample was incubated witheither 10 nM of recombinant Caspase-8, 1 ng of Granzyme B (Calbiochem),or 10 μM of Cytochrome C and 1 mM dATP, with or without various purifiedproteins in Caspase Buffer (1 mM EDTA, 0.1% Chaps and 10% Sucrose and 25mM HEPES pH 7.2). Then, 5 μl of reaction mixtures were incubated withfluorogenic caspase substrate acetyl-Asp-Glu-Val-Asp-aminofluorocoumarin(Ac-DEVD-AFC) (Calbiochem) in 100 μl Caspase buffer. Caspase activitywas assayed by using a LS50B fluorometric plate reader (Perkin-Elmer,Norwalk, Conn.) in the kinetic mode with excitation and emissionwavelength of 405 and 510 nm, respectively. Release of AFC from thesubstrate peptide was compared after 30 min incubation.

First, the effects of purified recombinant Survivin and XIAP onrecombinant active Caspase-3 were compared in vitro. In contrast toXIAP, which potently suppressed Caspase-3, Survivin displayed no abilityto suppress Caspase-3 protease activity, as measured by the hydrolysisof the fluorigenic peptide substrate Ac-DEVD-AFC (FIGS. 1 a and 1 b).Even at a 1000-fold molar excess of Survivin relative to activeCaspase-3, essentially no inhibition was observed. Similar results wereobtained using recombinant active Caspase-7 and -9. Again, XIAPinhibited, but Survivin did not.

Caspase activity in cell extracts was then measured, using exogenouslysupplied Cytochrome C to trigger activation of Caspases—a treatmentwhich is known to cause Cytochrome C-dependent oligomerization of theCaspase-activator, Apaf1, with Apaf1 then binding and activatingpro-Caspase-9, followed by cleavage and activation of downstreamprotease Caspase-3 (Zou, H. et al. 1999 J Biol Chem 274:11549-11556).When purified recombinant Survivin or XIAP was added to extracts priorto stimulation with Cytochrome C, Caspase activity in the cell lysateswas suppressed in a concentration-dependent manner by both proteins(FIGS. 1 c and 1 d). However, if Survivin or XIAP was added to extractsafter stimulation with Cytochrome C, then XIAP suppressed Caspaseactivity, whereas Survivin did not (FIG. 1 e and 1 f).

These results show that while Survivin is ineffective by itself atsuppressing Caspases, in collaboration with other proteins present incell lysates, Survivin can prevent Cytochrome C-mediated activation ofCaspases. However, unlike XIAP, Survivin does not appear to inhibitCaspases in cell lysates once they have been activated.

EXAMPLE 2 Identification of HBXIP as a Survivin Binding Partner

To identify potential partners of Survivin, we performed yeasttwo-hybrid screens of cDNA libraries using human Survivin protein asbait.

For library screening, a human Jurkat T cell cDNA library in pJG4-5 andthe EGY48 strain of Saccharomyces cerevisiae (MAT, trp1, ura3, his,leu2:plexApo6-leu2) were used, as described (Matsuzawa, S. & Reed, J. C.2001 Mol Cell 7:915-926). A cDNA encoding full-length Survivin wascloned into the EcoRI and XhoI sites of the yeast two-hybrid vector,pGilda (Clontech, Palo Alto, Calif.), which produces fusion proteinswith a LexA DNA-binding domain. Specificity of interactions wasconfirmed by mating experiments using a panel of yeast containingvarious control bait-plasmids (pGilda-Bax, -Fas, -caspase-9), and byre-transformation experiments (see FIG. 7).

From a pool of 64 candidate clones, 17 were found to represent cDNAsencoding the HBXIP protein, with all clones encoding the full-lengthprotein (see FIG. 7).

EXAMPLE 3 Cloning of Survivin, HBXIP and HBX and Production ofRecombinant Proteins

cDNA cloning and plasmid construction. A cDNA encoding human HBXIP wasgenerated by reverse transcription from Jurkat T cell mRNA usingSuperScript II (Gibco, Rockville, Md.), followed by the amplificationusing the Expand High Fidelity PCR system (Roche, Mannheim, Germany) andoligonucleotide primers as follows: 5′-GACGAATTCATGGAGGCGACCTTGGAGCA-3′(forward) (SEQ ID NO: 5) and 5′-GATCTCGAGTCAAGAGGCCATTTTGTGCA-3′(reverse) (SEQ ID NO: 6). The resultant cDNA fragments were ligated intothe plasmid pcDNA3-FLAG for mammalian expression (Matsuzawa, S. & Reed,J. C. 2001 Mol Cell 7:915-926), or into pET21d-N-His₆ and pGEX4T-1 forexpression in bacteria. Various fragments of Survivin cDNA were alsoPCR-amplified from pcDNA3-Survivin (Tamm, I. et al. 1998 Cancer Res58:5315-5320), and subcloned into pcDNA3 (Invitrogen, Carlsbad, Calif.).The gene encoding HBX was synthesized by PCR from DNA obtained frompatients with hepatocellular carcinoma, as described previously(Marusawa, H. et al. 2000 Hepatology 31:488-495).

Production of recombinant proteins and in vitro protein binding assays.Recombinant proteins were purified essentially as described (Deveraux,Q. L, & Reed, J. C. 1999 Genes Dev 13:239-252; Zou, H. et al. 1999 JBiol Chem 274:11549-11556). His₆-HBX protein was made using the RapidTranslation System (Roche), and 2 μl of synthesized reaction mixture wasused for Caspase assays. Purified GST-fusion protein or His₆-taggedproteins immobilized on glutathione-Sepharose beads or nickel beads,respectively, were incubated in 1% Triton-X 100/PBS for 1 h at 4° C.Then, the beads were washed with binding buffer (5 mM MgCl₂, 10%glycerol, 0.5 mg/ml BSA, 5 mM 2-mercaptoethanol and 50 mM Tris-Cl, pH7.5) and incubated overnight at 4° C. with various recombinant proteinsor in vitro translated-³⁵S-labeled proteins produced using TNT-coupledreticulocyte lysates (Promega, Madison, Wis.). Protein on beads werewashed four times in binding buffer, and bound proteins were eluted inSDS sample buffer, and subjected to SDS-PAGE, as described previously(Deveraux, Q. L. & Reed, J. C. 1999 Genes Dev 13:239-252).

EXAMPLE 4 Analysis of the binding of Survivin by HBXIP

HBXIP is a 91 amino-acid protein widely expressed in human tissues,whose function is presently unknown (Melegari, M. et al. 1998 J Virol72:1737-1743). The in vitro binding experiments were performed, in whicheither purified recombinant Survivin (untagged), which had been producedin bacteria as a Glutathione S-Transferase (GST) fusion protein and thendigested by thrombin to release Survivin, or GST-XIAP was incubated withHis₆-HBXIP, His₆-TRAF3 or SMAC-His₆ immobilized on nickel beads. Boundproteins were analyzed by immunoblotting using anti-Survivin (upperpanel) or anti-XIAP (middle panel) antisera. His₆-tagged-proteins werealso analyzed by SDS-PAGE/immunoblotting using anti-His₆ antibody (lowerpanel). These experiments confirmed the direct association of Survivinand HBXIP.

Gel-filtration chromatography. Recombinant pro-Caspase-9 (2 μg) wasincubated in the presence or absence of 5 μg Survivin and 5 μg HBXIP at30° C. for 30 min in 20 μl of buffer A. Then, 2 μg purified Apaf1, 200μM dATP, and 600 nM Cytochrome C was added to total volume of 55 μl.After incubation at 30° C. for 30 min, proteins were size-fractionatedthrough a Superdex-200 column (Amersham Biosciences, Piscataway, N.J.),collecting 100 μl fractions, and 20 μl of each fraction was subjected toSDS-PAGE, then blotted with anti-HBXIP, anti-Survivin, anti-Apaf1, andanti-Caspase-9 antibodies. Alternatively, fractions were incubated withGST-HBXIP, and proteins were recovered on glutathione-Sepharose.

To confirm that Survivin and HBXIP form stable complexes,gel-chromatography experiments were performed in which recombinantpurified Survivin, His-HBXIP, or the combination of these proteins wasanalyzed (FIGS. 2 b and 2 c). Purified Survivin alone (FIG. 2 b, upperpanel), His₆-HBXIP alone (FIG. 2 b, lower panel) or a combination ofSurvivin and His₆-HBXIP (FIG. 2 c) was subjected to gel-filtrationchromatography and eluted fractions were analyzed bySDS/PAGE/immunoblotting using anti-Survivin or anti-HBXIP polyclonalantibodies. Additionally, Ni-resin was added to fractions collected in(FIG. 2 c) to recover His₆-HBXIP, and associated Survivin was detectedby immunoblotting (“bound Survivin”) (FIG. 2 c, lower panel).

By itself, Survivin eluted at a size roughly consistent with the knowndimeric form of this protein. His₆-HBXIP, by itself, eluted at a sizecorresponding roughly to a trimer. When combined, however, some of theSurvivin and HBXIP was shifted to higher molecular weight fractions(FIG. 2 c). Moreover, recovering His₆-HBXIP from these slower-elutingfractions using nickel-Sepharose demonstrated the presence of associatedSurvivin. Thus, HBXIP and Survivin directly bind, forming complexes.

Scatchard analysis showed that HBXIP binds Survivin with sub-micromolaraffinity (K_(D)˜460 nM) (FIG. 2 d), which supports the idea that thisinteraction is sufficiently tight to be physiologically relevant.

To map the region of Survivin required for interaction with HBXIP, aseries of fragments of Survivin were prepared by in vitro-translation inthe presence of ³⁵S-L-methionine, and assayed for their ability to bindpurified GST-HBXIP. Survivin fragments were incubated with GST-CD40(cytosolic domain) control protein or GST-HBXIP immobilized onglutathione-Sepharose. Bound proteins were analyzed by autoradiography.Survivin is the smallest of the mammalian IAPs, containing only a singleBIR (residues 15-88), followed by a dimerization domain (residues89-126), and an α-helical region (residues 126-142) that mediatesassociation of this protein with mitotic structures in dividing cells(Verdecia, M. A. et al. 2000 Nature Struct Biol 7:602-608). Fragments ofSurvivin retaining the BIR domain bound HBXIP in vitro, while fragmentslacking the BIR did not (FIG. 2 e). Moreover, a fragment of Survivinrepresenting only the BIR domain bound HBXIP, demonstrating that the BIRof Survivin is necessary and sufficient for HBXIP binding in vitro.

EXAMPLE 5 Confirmation of Survivin binding to HBXIP byCo-Immunoprecipitation

For co-immunoprecipitation and immunodepletion assays, cells werecultured with 20 μM MG-132 (Calbiochem) for 8 h before lysing in IPbuffer (0.5% NP-40, 1 mM EDTA, 135 mM NaCl and 20 mM Tris-Cl, pH 7.5)containing protease inhibitors (Complete, Roche), and then incubatedwith primary antibody immobilized on recombinant protein G-Sepharose 4B(Zymed, South San Francisco, Calif.) at 4° C. overnight with constantrotation. Immunoprecipitates were washed with IP buffer four times andsuspend in SDS sample buffer, then boiled and analyzed bySDS-PAGE/immunoblotting. Immunodepletion analysis was performed asdescribed using 5 μl of anti-Survivin antiserum or preimmune serumconjugated with 20 μl of protein A-Sepharose (Pathan, N. et al. 2001 JBiol Chem 276:32220-32229).

HBXIP binding to Survivin in cells was confirmed byco-immunoprecipitation assays, using epitope-tagged proteins produced inHEK293T cells by transient transfection (FIG. 2 f). 293T cells weretransiently transfected with plasmids encoding myc-tagged Survivin ormyc-XIAP, together with FLAG-HBXIP or FLAG-SIP (control). Lysates weresubjected to immunoprecipitation using anti-FLAG epitope antibody.Immunoprecipitates were analyzed by immunoblotting using anti-mycantibody (upper panel). Lysates were also blotted by anti-myc (middlepanel) or anti-FLAG antibodies (lower panel). Moreover, HBXIP bound toSurvivin but not to XIAP, confirming a specific interaction. Binding ofendogenous HBXIP to endogenous Survivin was also detected byco-immunoprecipitation assays, using anti-Survivin antibody toimmunoprecipitate Survivin, followed by immunoblot analysis of theresulting immune-complexes using anti-HBXIP antiserum. In thisexperiment lysates from untransfected HepG2 cells were prepared forimmunoprecipitation with anti-Survivin antisera, followed by blottingwith anti-HBXIP or anti-Survivin antibodies. (FIG. 2 g).

EXAMPLE 6 Antibodies Specific to Survivin, HBXIP, Pro-Caspase-9,Pro-Caspase-3, and XIAP

Polyclonal antisera specific for HBXIP were generated in rabbits usingpurified recombinant His₆-HBXIP as an immunogen. Rabbit polyclonalantibody against pro-Caspase-9, pro-Caspase-3, XIAP and Survivin havebeen described (Krajewski, S. et al. 1999 PNAS USA 96:5752-5757; Tamm,I. et al. 1998 Cancer Res 58:5315-5320; Krajewska, M. et al. 1997 CancerRes 57:1605-1613). Rabbit polyclonal antibody recognizing human Apaf1was purchased from Cayman Chemical Company (Ann Arbor, Mich.). Mousemonoclonal antibodies against active Caspase-9 and Cytochrome C werepurchased from Pharmingen (San Diego, Calif.). Rabbit polyclonalanti-HBX antibody was generously provided by Robert J. Schneider (NewYork University Medical School).

EXAMPLE 7 Subcellular Localization of Survivin and HBXIP

Subcellular fractionation experiments were performed to determinewhether Survivin and HBXIP reside in the same cellular compartment invivo. Cells were lysed in hypotonic buffer (10 mM Tris-HCl pH 7.5, 10 mMNaCl, 1 mM EDTA, protease inhibitors Complete, Roche) and centrifuged at700 g for 5 min to obtain nuclear pellets. The resulting supernatantswere further centrifuged at 100,000 g for 60 min to obtain membranes(pellet) and soluble cytosolic (supernatant) fractions. Lysates fromunfractionated 293 cells (T) and from subcellular fractions (M;membrane, C; cytoplasmic, N; nuclear), normalized for cell-equivalents,were analyzed by immunoblotting using anti-HBXIP and anti-Survivin.Blotting using anti-HSP60, anti-Caspase-3 and anti-PARP antibodies wasalso performed as markers for membrane (mitochondrial), cytosolic, andnuclear proteins, respectively (FIG. 2 h).

Both HBXIP and Survivin were found predominantly in the cytosolicfraction, though some Survivin was also seen in the nuclear fraction(FIG. 2 h).

EXAMPLE 8 HBXIP Collaborates with Survivin in Suppressing Caspase-9Activation

To explore the functional significance of the interaction of HBXIP withSurvivin, the effects of recombinant HBXIP alone and in combination withSurvivin on activation of Caspases in cell lysates stimulated withCytochrome C were evaluated. At concentrations below 200 nM, addition ofeither HBXIP or Survivin individually to cell lysates only slightlysuppressed the generation of Caspase protease activity, as measured bythe hydrolysis of Ac-DEVD-AFC (FIG. 3 a). In contrast, the combinationof HBXIP and Survivin nearly completely suppressed Cytochrome C-mediatedactivation of Caspases, implying functional synergy of these proteins.However, if Survivin and HBXIP were added after Cytochrome Cstimulation, Caspase-activity was not suppressed, implying that theseproteins block the activation event but do not suppress Caspases oncethey have been activated. Replacing either Survivin or HBXIP withvarious control proteins (e.g., GST; GST-CD40; His₆-TRAF3) failed tosuppress Caspase activity, confirming the specificity of these results.

In contrast to Cytochrome C, addition of the combination of Survivin andHBXIP to cell lysates did not significantly inhibit Caspase activationinduced by addition of either purified recombinant active Caspase-8(FIG. 3 b) or Granzyme B (FIG. 3 c). By comparison, XIAP completelysuppressed effector Caspase activity in these lysates. These findingstherefore show that Survivin selectively inhibits Caspase activation viathe Cytochrome C pathway, whereas XIAP has broader activity.

The ability of the combination of HBXIP and Survivin to selectivelysuppress Caspase activation induced by Cytochrome C prompted furtherexploration into the effects of these proteins on Apaf1-mediatedactivation of pro-Caspase-9, the apical protease in the Cytochrome Cpathway for apoptosis (Li, P. et al. 1997 Cell 91:479-489). For theseexperiments, recombinant Apaf1 and pro-Caspase were produced in andpurified from insect cells, as described (Zou, H. et al. 1999 J BiolChem 274:11549-11556). After addition of Cytochrome C and dATP to induceApaf1 oligomerization, Caspase-9 activity was measured by cleavage ofthe fluorigenic substrate Ac-LEHD-AFC (FIG. 3 d) and pro-Caspase-9processing was monitored by immunoblotting (FIG. 3 e). Addition ofeither recombinant purified Survivin or HBXIP individually wasineffective at blocking Apaf1-mediated activation and proteolyticprocessing of pro-Caspase-9 in vitro. In contrast, the combination ofSurvivin and HBXIP effectively suppressed generation of Caspase-9protease activity (FIG. 3 d), and also reduced proteolytic processing of50 kDa pro-Caspase-9 into 35 kDa large and ˜12 kDa small subunitstypical of the active protease (FIG. 3 e). This effect of combiningHBXIP and Survivin was not merely due to the presence of more protein inthe reactions, because substituting various control proteins for eitherHBXIP or Survivin (e.g. GST; GST-CD40; His₆-TRAF3) failed to inhibitApaf1-mediated activation of pro-Caspase-9.

EXAMPLE 9 HBXIP/Survivin Complexes can Bind Pro-Caspase-9, Preventingits Activation by Apaf1

To explore the mechanism by which the combination of Survivin and HBXIPsuppresses pro-Caspase-9 activation, testing was performed to determinewhether these proteins can bind pro-Caspase-9 in vitro. For theseexperiments, GST-Survivin, GST-CD40 or GST-HBXIP with or withoutpurified Survivin (untagged) was incubated with His₆-pro-Caspase-9, andthe HBXIP bound proteins were analyzed by immunoblotting using ananti-Caspase-9 antiserum (Krajewski, S. et al. 1999 PNAS USA96:5752-5757). For comparative purposes, similar experiments were alsoperformed using recombinant purified active His₆-Caspase-9 (lacking CARDdomain) (Stennicke, H. R. et al. 1997 J Biol Chem 274:8359-8362) andHis₆-pro-Caspase-3 (Stennicke, H. R. & Salvesen, G. S. 1997 J Biol Chem272:25719-25723). GST-fusion proteins were recovered usingglutathione-Sepharose and bound proteins were detected by immunoblottingusing anti-Caspase-9 or anti-Caspase-3 antisera. An equivalent amount ofproteins was loaded directly in gels as a control (“input”). In theabsence of Survivin, GST-HBXIP failed to “pull-down” Caspases (FIG. 4a). However, when Survivin was included, then pro-Caspase-9 waspulled-down with GST-HBXIP, but not with control GST-CD40 protein.GST-HBXIP also pulled-down active Caspase-9, in the presence but not theabsence of Survivin, but the proportion of active Caspase-9 thatassociated with GST-HBXIP under these conditions was far less thanpro-Caspase-9. No association with Caspase-3 was found. Also, whenSurvivin was used alone (as a GST-fusion protein) for pull-down assays,no binding to Caspases was detected (FIG. 4 a, lane 3), providingfurther evidence that Survivin cannot bind Caspases by itself. Takentogether with the data on Caspase-9 activity, it was shown thatHBXIP/Survivin complexes can bind pro-Caspase-9, preventing itsactivation by Apaf1.

Given that HBXIP/Survivin complexes bind pro-Caspase-9, further analysiswas performed to assess whether these proteins interfere withassociation of pro-Caspase-9 with active Apaf1. For these experiments,recombinant purified His₆-Apaf1 was incubated with ³⁵S-labeledpro-Caspase-9 (produced by in vitro translation), Cytochrome C and dATP,in the presence or absence of Survivin, HBXIP, or the combination ofthese proteins. Then, His₆-Apaf1 was recovered on Ni-chelation resin,and associated proteins were analyzed by autoradiography (Caspase-9)(FIG. 4 b) or by immunoblotting using anti-Cytochrome C antibody (FIG. 4c, FIG. 4 d). In the absence of Survivin and HBXIP, full-length ˜50 kDapro-Caspase-9 as well as the 35-37 kDa large and 12 kDa small subunitsof processed Caspase-9 were recovered with His₆-Apaf1 on Ni-resin (FIG.4 b). In contrast, when pro-Caspase-9 was pre-incubated with thecombination of HBXIP and Survivin before the introduction of His₆-Apaf1,the amount of Pro-Caspase and processed Caspase-9 recovered withHis₆-Apaf1 on Ni-resin was significantly reduced, indicating thatHBXIP-Survivin complexes prevented recruitment of pro-Caspase-9 toCytochrome C-activated Apaf1 (FIG. 4 b). When various control proteinswere substituted for either Survivin or HBXIP, then pro-Caspase-9association with Apaf1 was not blocked. Also, Survivin/HBXIP complexesdid not interfere with binding of Cytochrome C to Apaf1 (FIG. 4 b),demonstrating a specific effect on recruitment of pro-Caspase-9.

EXAMPLE 10 Survivin/HBXIP Inhibits Apoptosome Formation and Activity

The multiprotein complex containing Apaf1, Cytochrome C, and Caspase-9is called the “apoptosome” (Zou, H. et al. 1999 J Biol Chem274:11549-11556). Assays of apoptosome activity were conductedessentially as described, using proteins produced in and purified frombaculovirus-infected insect cells (Zou, H. et al. 1999 J Biol Chem274:11549-11556). Recombinant pro-Caspase-9 (4 nM) was incubated withpurified Survivin (40 nM), HBXIP (40 nM), the combination of theseproteins, or equivalent amounts of various control proteins such as CD40or TRAF3 at 30° C. for 10 min. Then, 4 nM recombinant Apaf1 was added,followed by 200 μM dATP, 600 nM Cytochrome C, and 10 μM of recombinantpro-Caspase-3 in a total of 40 μl of buffer A, as described above. Afterincubation, 1601 of Caspase buffer containing Ac-DEVD-AFC (100 μM finalconcentration) was added. Alternatively, for measurements of Caspase-9activity, 200 nM of pro-Caspase-9 was incubated with 100 nM of Apaf1with 2 μM of various control or specific recombinant proteins under thesame conditions, then Ac-Leu-Glu-His-Asp-AFC (Ac-LEHD-AFC, Calbiochem)fluorigenic substrate was added.

To further address the mechanism by which Survivin/HBXIP complexesinhibit Apaf1-induced activation of pro-Caspase-9, apoptosome complexeswere subjected to gel-filtration chromatography, analyzing columnfractions by SDS-PAGE/immunoblotting using antibodies recognizing Apaf1or Caspase-9 (FIG. 4 c). In the absence of Survivin and HBXIP, two peaksof Apaf1 were detected, and Caspase-9 co-eluted with the larger or thesecomplexes (FIG. 4 c), consistent with prior reports (Zou, H. et al. 1999J Biol Chem 274:11549-11556). In contrast, when apoptosome assembly wasinduced in the presence of Survivin and HBXIP, and then gel-filtrationchromatography analysis was performed, very little Caspase-9 co-elutedwith Apaf1. Also, more of the Caspase-9 was present in the pro-enzymeform (˜50-kDa as opposed to 35 kDa) when HBXIP and Survivin wereincluded. Moreover, the column fractions in which the majority of thepro-Caspase-9 eluted also contained Survivin and HBXIP (FIG. 4 d).

In addition, column fractions were monitored for Caspase-9 activity,using an assay in which the Caspase-9 substrate, pro-Caspase-3, wasadded and then Caspase-3 activity was measured by AC-DEVD-AFChydrolysis. Comparisons of apoptosome-associated Caspase-9 activitydemonstrated markedly reduced Caspase-9 activity when Survivin and HBXIPwere included (FIG. 4 e). It is therefore concluded that the combinationof HBXIP and Survivin reduces pro-Caspase-9 activation by interferingwith apoptosome assembly.

If Survivin and HBXIP prevent pro-Caspase-9 activation by Apaf1, thenone would predict that co-expressing Survivin and HBXIP in cells wouldprovide protection from apoptotic stimuli that operate through aCaspase-9-dependent pathway, but not from stimuli that induce apoptosisthrough other routes. Compared were the effects of transfecting plasmidsencoding Survivin, HBXIP, or the combination of these proteins onapoptosis induced by Staurosporine and anti-Fas antibody, stimuli thatoperate through Caspase-9-dependent and -independent pathways,respectively (Salvesen, G. S. 2002 Cell Death Differ 9:3-5).

Cell lines were cultured in DMEM with 10% fetal calf serum, 1 mML-glutamine, and antibiotics. Cells were transfected with variousplasmids in combination with pEGFP (Clontech) using Fugene-6transfection reagent (Roche). After culturing 1.5 days, apoptosis wasinduced by anti-Fas monoclonal antibody CH-11 (500 ng/ml; MBL, Nagoya,Japan), or Staurosporine (100 nM). Both floating and attached cells werecollected 24 h after apoptosis induction, and analyzed by 1 μg/ml of4′,6-diamidino-2′-phenylindole dihydrochloride (DAPI) staining forassessing nuclear morphology. The percentage of apoptotic cells revealedby DAPI staining was determined by fluorescence microscopy, counting aminimum of 200 GFP-positive cells.

In addition, wild-type HBXIP was compared with a mutant containing onlythe first 40 amino-acids of HBXIP protein, del-HBXIP (1-40), that weempirically determined is incapable of binding Survivin (FIG. 4 f upperpanel). At the plasmid concentrations used, either Survivin or HBXIPonly slightly suppressed apoptosis in transfected HT1080 cells (FIG. 4f, lower panel). In comparison, Bcl-XL, which inhibits Cytochrome Crelease from mitochondria (Yang, J. et al. 1997 Science 275:1129-1132)and CrmA, which inhibits Caspase-8 (Zhou, Q. et a/l. 1997 J Biol Chem272:7797-7800) suppressed apoptosis induced by Staurosporine andanti-Fas antibody, respectively, serving as positive-controls for theseassays. However, when Survivin and HBXIP were co-expressed in HT1080cells, then apoptosis induced by Staurosporine, but not Fas, wassuppressed (FIG. 4 f). Immunoblot analysis of transfected cells, whichwere treated with a broad-spectrum Caspase inhibitor (zVAD-fmk) toprevent apoptosis, confirmed production of all proteins and demonstratedcomparable levels of HBXIP and del-HBXIP (1-40).

Taken together, these data demonstrate a collaborative role of Survivinand HBXIP in selectively suppressing the Caspase-9-dependent pathway forapoptosis, consistent with the idea that these proteins interfere withpro-Caspase-9 activation.

EXAMPLE 11 Endogenous HBXIP and Survivin Regulate Caspase Activation inCancer Cells

To explore the role of HBXIP in the pathogenesis of human liver diseaseassociated with HBV infection, the expression of this protein was firstanalyzed by immunoblotting in primary hepatocellular carcinoma tissues,as well as non-cancerous regions of the same livers of patients withchronic HBV infection. The non-cancerous regions of these liver tissuesincluded two cases with liver cirrhosis (Case #1 and 2) and one withchronic hepatitis (Case #3). As a control for non-HBV infection, normalregions of hepatic tissue from patients with metastatic colon cancerwere also examined. Levels of HBXIP protein were elevated in bothcancerous and non-cancerous liver tissue of patients with chronic HBVinfection, compared to hepatic tissue of patients without a history ofHBV infection (FIG. 5 a). Based on normalization to α-tubulin levels,HBXIP levels were determined to be 2- to 7-fold higher in the tissues ofpatients with chronic HBV infection compared to non-infected liver,based on scanning-densitometry analysis of immunoblots. Interestingly,probing the same blots with anti-Survivin antibody revealed higherlevels of Survivin protein in tumor tissue compared non-cancerousregions of the same livers in all 3 specimens examined (FIG. 5 a).

Since the combination of Survivin and HBXIP interferes with CytochromeC-mediated activation of pro-Caspase-9 (see above), it was determinedwhether a correlation exists between elevated expression of theseproteins in HBV-associated hepatocellular carcinomas and resistance toCytochrome C-induced Caspase activation. Accordingly, lysates from theliver tissues were normalized for total protein content, and thenincubated with Cytochrome C and dATP to induce Apaf1-mediated activationof Caspases. Compared to non-infected liver tissue, CytochromeC-inducible Caspase activity was significantly reduced in lysates ofboth cancerous (p<0.01) and non-cancerous (p<0.01) liver tissue derivedfrom patients with chronic HBV infection (FIG. 5 b, left panel), withinduction of DEVD-cleaving Caspase activity more depressed in canceroustissues than non-cancerous tissues from HBV-infected individuals(p<0.01). In contrast, no significant difference in Caspase activityinduced by exogenously added Granzyme B was found, comparing normal,HBV-infected, and cancer tissues (FIG. 5 b, right panel). Thus,HBV-associated elevations in expression of HBXIP correlate withselective resistance to Cytochrome c-mediated activation of Caspases inextracts from primary patient tissues.

EXAMPLE 12 Reduction of HBXIP Expression by siRNA RelievesSurvivin-Mediated Suppression of Apoptosis

To determine whether HBXIP is necessary for the inhibitory effect ofSurvivin, small-interfering RNA (siRNA) were used to reduce expressionof endogenous HBXIP in HeLa cells. siRNA duplexes composed of21-nucleotide sense and antisense strands were synthesized by DharmaconResearch (Lafayette, Colo.). RNA oligonucleotides used for targetingHBXIP in this study were: HBXIP-S1,5′-GCAGCUAAGCUAACCUCUGTT-3′ (sense)(SEQ ID NO: 7), and HBXIP-AS1 5′-TTCGUCGAUUCGAUUGGAGAC-3′ (antisense)(SEQ ID NO: 8). HeLa cells were plated in 6 cm wells at 2.5×10⁵ cellsper well 24 h before transfection. 20 μM siRNA in 2511 of Oligofectaminereagent (Invitrogen) was incubated in serum-free Opti-MEM medium for 20min, then the transfection mixture was added to cells, incubated at 37°C. for 4 h, followed by addition of fresh medium containing serum. At 36hours after transfection, cells were analyzed for apoptosis or lysed forimmunoblotting.

Transfection of HBXIP-specific but not control double-strand syntheticRNAs reduced levels of endogenous HBXIP protein in HeLa cells, which wassustained for at least 3 days, as determined by immunoblot analysis(FIG. 5 c). It was also confirmed that the levels of other proteinsrequired for Cytochrome C responsiveness (Apaf1, pro-Caspase-9,pro-Caspase-3) were not affected by treatment with HBXIP-siRNA. Caspaseactivity induced by Cytochrome C in cell lysates derived fromsiRNA-treated cells was then measured. Extracts prepared from HeLa cellsafter treatment with HBXIP-siRNA or control-RNA were incubated withCytochrome c and dATP, in the presence or absence recombinant Survivin(left panel) or XIAP (right panel), and Caspase activity was measuredbased on release of AFC from Ac-DEVD-AFC substrate (mean ±SE; n=3determinations) (FIG. 5 d). Cytochrome C (with dATP) was more effectiveat activating Caspases in cell extracts treated with HBXIP-siRNAcompared to control RNA-treated cells, confirming a role for HBXIP as anendogenous antagonist of this Caspase activation pathway. In controlextracts, addition of recombinant purified Survivin suppressedCytochrome C-mediated activation of Caspases. In contrast, Survivinfailed to suppress Caspase activation in extracts in which HBXIPexpression was knocked-down (FIG. 5 d left panel). By comparison,siRNA-mediated knock-down of HBXIP did not affect the inhibitory effectof recombinant XIAP on Cytochrome C-mediated Caspase activation (FIG. 5d right panel), demonstrating the specificity of these results.

Similar conclusions were reached from experiments using intact cells inwhich endogenous HBXIP expression was knocked-down by siRNA, showingthat more apoptosis was induced by agents that triggerCaspase-9-dependent apoptosis, such as DNA-damaging agent VP16(etoposide) and kinase-inhibitor Staurosporine (STS), when HBXIP levelswere reduced (FIG. 5 e). In these experiments the percentage ofapoptotic cells (mean ±SE; n=3) was determined by DAPI-stainingfollowing culture of control-RNA- or HBXIP siRNA-transfected HeLa cellswith Etoposide (VP-16) or Staurosporine (STS).

EXAMPLE 13 HBX Protein Collaborates with HBXIP in Suppressing CaspaseActivation

The viral oncoprotein HBX is encoded in the HBV genome, and has beenimplicated in hepatocellular carcinogenesis through uncertain mechanisms(reviewed in Murakami, S. 2001 J Gastroenterol 36:651-660). In vitroprotein binding assays were performed in which recombinant His₆-HBX wasincubated with GST-HBXIP, GST-Survivin, or GST-CD40 (control)immobilized on glutathione-Sepharose. Bound proteins were analyzed byimmunoblotting using anti-HBX antibody. This confirmed previouslyreported observations that HBX associates with HBXIP (FIG. 6 a). HBX, incontrast, did not bind Survivin.

The ability of HBX to associate with HBXIP raised the question ofwhether this viral oncogenic protein could effect Caspases. Sinceattempts to produce soluble recombinant HBX in bacteria failed, in vitrotranslation using bacterial extracts was used for production of thisviral protein. His₆-HBX or control [CTR] proteins, purified His₆-HBXIP,purified Survivin, or various combinations of these proteins were addedto 293 cell extracts normalizing all samples for total protein addedusing control recombinant proteins, then Cytochrome c and dATP wereadded, and Caspase activity was measured 30 min later based onhydrolysis of Ac-DEVD-AFC. Release from fluorogenic AFC from AC-DEVD-AFCwas measured continuously. Addition of HBX protein to lysates preparedfrom HepG2 hepatocellular cancer cells suppressed Caspases activationinduced by Cytochrome C and dATP, while control proteins prepared in thesame manner had little effect (FIG. 6 b). In addition, this viralprotein augmented the inhibitory effect of recombinant purified HBXIPand Survivin on Cytochrome C-mediated Caspase activation (FIG. 6 c),prompting examination into whether HBX protein associates withHBXIP/Survivin complexes.

For this purpose, co-immunoprecipitation assays were carried out usingepitope-tagged HBX, HBXIP and Survivin expressed by transienttransfection in HEK293T cells. Since HBX does not directly bind Survivin(FIG. 6 a), it was reasoned that over-expressing HBXIP would increasethe amounts of Survivin immunoprecipitated with HBX by bridging thesetwo proteins together. HEK293 cells were transiently transfected withplasmids encoding FLAG-tagged-HBX or FLAG-SIP (as a control) togetherwith Myc-Survivin or HA-HBXIP or control plasmid. Lysates were subjectedto immunoprecipitation using anti-FLAG epitope antibody, demonstratingincreased association of Survivin with HBX when HBXIP was co-expressed(compare last two lanes at right). Immunoprecipitates were analyzed byimmunoblotting using anti-Survivin antibody (upper panel). Lysates werealso analyzed by immunoblotting using anti-HBX (middle panel) or anti-HAantibodies (lower panel), confirming production of the intendedproteins. Indeed, when HBX and Survivin were co-expressed with HBXIP,considerably more Survivin was associated with HBX-immunoprecipitates(FIG. 6 d). These data therefore are consistent with the idea that HBX,HBXIP, and Survivin form complexes.

To address whether Survivin/HBXIP complexes were still capable ofbinding pro-Caspase-9 in the presence of HBX, we performed in vitroprotein interaction assays, examining the association of pro-Caspase-9with GST-HBXIP when Survivin, HBX, or both proteins were added.His₆-pro-Caspase-9 was incubated with GST-HBXIP(+) or GST-CD40 controlprotein (−), in the presence or absence of His₆-HBX and untaggedSurvivin. GST-fusion proteins were recovered on glutathione-Sepharoseand bound proteins were detected by immunoblotting using anti-Caspase-9,anti-Survivin, or anti-HBX antisera. As shown above, GST-HBXIPpulled-down pro-Caspase-9 when Survivin was included in the bindingassays (FIG. 6 e lane 3). Addition of HBX did not impair pro-Caspase-9association with GST-HBXIP, and instead increased pro-Caspase-9 bindingslightly (FIG. 6 e lane 4). Thus, rather than competing for binding toHBXIP, the HBX protein appears to form complexes with HBXIP in a mannerthat does not preclude simultaneous association with Survivin andpro-Caspase-9, and which may even enhance formation of multiproteincomplexes containing these proteins.

To further explore whether HBX suppresses Caspase activity through aSurvivin-dependent mechanism, endogenous Survivin was immunodepletedfrom HepG2 cell lysates using anti-Survivin antisera or preimmune serum(CTR) and then equivalent amounts analyzed by immunoblotting usinganti-Survivin antibody (top panel). Further, equivalent volumes ofextracts were analyzed for Caspase activity based on Ac-DEVD-AFCcleavage, where lysates were incubated with recombinant HBX (+) orcontrol protein (−) prior to stimulation with Cytochrome c and dATP. Inextracts subjected to mock immunodepletion, HBX suppressed Caspaseactivation by roughly half (FIG. 6 f). In contrast, when Survivin wasimmunodepleted, it was found that Cytochrome C was more potent atactivating Caspases and that HBX had little inhibitory activity (FIG.6). Thus, HBX fails to inhibit Caspases in the absence of Survivin.

EXAMPLE 14 Pathways Leading to Cell Survival and Cell Death

In summary, it was discovered that Survivin requires an additionalpartner protein in its mechanism of Caspase inhibition that results ininhibition of apoptosis. Using yeast two-hybrid screens of cDNAlibraries, HBXIP was identified as a candidate Survivin-binding protein,and the association of HBXIP with Survivin was confirmed in vitro and incells by several methods. Importantly, using purified components, it wasfound that the combination of Survivin and HBXIP was required forbinding and preventing Apaf1-mediated activation of pro-Caspase-9,whereas neither protein individually was adequate. Moreover, thecombination of Survivin and HBXIP, but neither alone, reducedrecruitment of pro-Caspase-9 to Cytochrome c-activated Apaf1. Consistentwith the discovery that Survivin requires HBXIP for itsCaspase-inhibitory function, it was shown that Survivin lost its abilityboth to suppress Cytochrome C-mediated activation of Caspases in cellextracts and to inhibit apoptosis when expressed in intact cells inwhich endogenous HBXIP had been largely eradicated by siRNA.

HBXIP was originally isolated as a human protein which binds the viraloncogenic protein, HBX, of the Hepatitis 13 Virus (HBV) (Melegari, M. etal. 1998 J Virol 72:1737-1743). HBXIP encodes a protein of 9,6-kDa witha putative leucine zipper motif. Expression of HBXIP mRNA has beendemonstrated in essentially all tissues examined to date, and is notlimited to the liver (Melegari, M. et al. 1998 J Virol 72:1737-1743). Inthe context of HBV-infection, HBXIP reportedly reduces viral replicationand abolishes the transactivation function of viral HBX protein(Melegari, M. et al. 1998 J Virol 72:1737-1743), however, little isknown about the physiological roles of HBXIP in human cells. Thecellular HBXIP protein is conserved in mice and rodents, suggesting anevolutionarily conserved function. Based on the data presented here,HBXIP is envisioned to be an anti-apoptotic co-factor of Survivin.Consistent with this idea, siRNA-mediated reductions in endogenous HBXIPlevels sensitized cells to apoptosis, while over-expression of HBXIPsuppressed apoptosis in collaboration with Survivin.

Up-regulation of HBXIP was found in both cancerous and non-malignantliver tissue of humans with chronic HBV-infection, compared to normalhepatic tissue. In this regard, chronic HBV infection is known to causepre-neoplastic changes in liver (Lok, A. S. 2000 J Hepatol 32:89-97),explaining the observed elevations in HBXIP in both non-malignant andtumor tissue of HBV-diseased livers.

It is estimated that more than 380 million HBV carriers are presentworldwide today, with chronic HBV infection representing a major globalpathogenic factor for development of hepatocellular carcinoma (Lok, A.S. 2000 J Hepatol 32:89-97). A crucial role of HBV inhepatocarcinogenesis is beyond doubt, while the mechanisms by which HBVcauses the transformation of hepatocytes remain unclear. The HBV genomeconsists in a 3.2 kbp circular double-stranded DNA molecule withoverlapping open reading frames (ORFs) encoding four viral proteins.Among them is HBX, a 154 amino-acid protein that has no recognizablecounterpart in humans or other mammalian species. HBX is essential forreplication of woodchuck HBV, and transgenic mice engineered to expressHBX have increased incidence of hepatocellular cancer, especially whenexposed to chemical carcinogens (Kim, C. M. et al. 1991 Nature351:317-320). Thus, HBX is a candidate transforming gene of HBV. Likemany viral oncoproteins, HBX is multifunctional protein. HBX exhibitseffects on gene transcription, cell proliferation, and apoptosis, andhas multiple putative cellular targets besides HBXIP (reviewed inMurakami, S. 2001 J Gastroenterol 36:651-660). The effects of HBX onapoptosis are controversial, with evidence suggestive either ofsuppression or promotion of apoptosis, depending on the cellular contextand stimulus. Among the reported molecular effects of HBX istranscription-independent suppression of Caspases (Gottlob, K. et al.1998 J Biol Chem 273:33347-33353), though many alternative mechanismshave also been proposed (reviewed in Murakami, S. 2001 J Gastroenterol36:651-660). These data demonstrate that HBX can associate indirectlywith Survivin, through HBXIP. Moreover, depletion of Survivin from cellextracts abolishes the ability of HBX to suppress Caspase activation invitro. Thus, HBX modulates apoptosis pathways through interactions withHBXIP/Survivin complexes.

The hepatitis B X-interacting protein (HBXIP) is a target for cancertherapy. The interaction of HBXIP with Survivin can be suppressed inneoplastic cells by downregulating the HBXIP expression using siRNA orantisense. The level of HBXIP in neoplastic cells can be reduced in thepresence of HBXIP- or Survivin-specific antibodies. The interaction ofHBXIP with Survivin may also be inhibited by the use of specificinhibitors, molecular decoys, or the like.

While the present invention has been described in some detail forpurposes of clarity and understanding, one skilled in the art willappreciate that various changes in form and detail can be made withoutdeparting from the true scope of the invention. All figures, tables, andappendices, as well as patents, applications, and publications, referredto above, are hereby incorporated by reference in their entireties.

1. An isolated compound that inhibits interaction of Survivin withhepatitis B X-interacting protein (HBXIP), wherein said compoundcomprises an antisense nucleic acid molecule or siRNA molecule thatinhibits expression of HBXIP.
 2. The isolated compound of claim 1,wherein said compound comprises about 15 to about 30 nucleotides.
 3. Theisolated compound of claim 1, wherein said compound comprises a modifiedbackbone.
 4. The isolated compound of claim 3, wherein said modifiedbackbone comprises phosphorothioates.
 5. The isolated compound of claim1, wherein said compound has greater than 90% identity with a nucleicacid molecule encoding said HBXIP.
 6. The isolated compound of claim 1,wherein said HBXIP is encoded by the nucleic acid sequence shown in SEQID NO:
 3. 7. The isolated compound of claim 1, wherein said compoundcomprises an antisense molecule.
 8. The isolated compound of claim 1,wherein said compound comprises an siRNA molecule.
 9. The isolatedcompound of claim 1, wherein said compound comprises one or moresubstituted sugar moieties.
 10. A pharmaceutical composition comprisinga compound that inhibits interaction of Survivin with hepatitis BX-interacting protein (HBXIP), wherein said compound comprises anantisense nucleic acid molecule or siRNA molecule and a pharmaceuticallyacceptable carrier.
 11. The pharmaceutical composition of claim 10,wherein said compound comprises about 15 to about 30 nucleotides. 12.The pharmaceutical composition of claim 10, wherein said compoundcomprises a modified backbone.
 13. The isolated compound of claim 12,wherein said modified backbone comprises phosphorothioates.
 14. Thepharmaceutical composition of claim 10, wherein said compound hasgreater than 90% identity with a nucleic acid molecule encoding saidHBXIP.
 15. The pharmaceutical composition of claim 10, wherein saidHBXIP is encoded by the nucleic acid sequence shown in SEQ ID NO:
 3. 16.The pharmaceutical composition of claim 10, wherein said compoundcomprises an antisense molecule.
 17. The pharmaceutical composition ofclaim 10, wherein said compound comprises an siRNA molecule.
 18. Theisolated compound of claim 10, wherein said compound comprises one ormore substituted sugar moieties.